Inhibition and Structure of Toxoplasma gondii Purine NucleosidePhosphorylase

Teraya M. Donaldson,a María B. Cassera,b Meng-Chiao Ho,b Chenyang Zhan,b Emilio F. Merino,b Gary B. Evans,c Peter C. Tyler,c

Steven C. Almo,b Vern L. Schramm,b Kami Kima,d,e

Departments of Microbiology and Immunology,a Biochemistry,b Pathology,d and Medicine,e Albert Einstein College of Medicine, Yeshiva University, Bronx, New York, USA;Carbohydrate Chemistry Group, Callaghan Innovation, Lower Hutt, New Zealandc

The intracellular pathogen Toxoplasma gondii is a purine auxotroph that relies on purine salvage for proliferation. We have op-timized T. gondii purine nucleoside phosphorylase (TgPNP) stability and crystallized TgPNP with phosphate and immucillin-H,a transition-state analogue that has high affinity for the enzyme. Immucillin-H bound to TgPNP with a dissociation constant of370 pM, the highest affinity of 11 immucillins selected to probe the catalytic site. The specificity for transition-state analoguesindicated an early dissociative transition state for TgPNP. Compared to Plasmodium falciparum PNP, large substituents sur-rounding the 5=-hydroxyl group of inhibitors demonstrate reduced capacity for TgPNP inhibition. Catalytic discriminationagainst large 5= groups is consistent with the inability of TgPNP to catalyze the phosphorolysis of 5=-methylthioinosine to hypo-xanthine. In contrast to mammalian PNP, the 2=-hydroxyl group is crucial for inhibitor binding in the catalytic site of TgPNP.This first crystal structure of TgPNP describes the basis for discrimination against 5=-methylthioinosine and similarly 5=-hy-droxy-substituted immucillins; structural differences reflect the unique adaptations of purine salvage pathways of Apicomplexa.

Toxoplasma gondii, the etiologic agent of toxoplasmosis, is anopportunistic pathogen that is widespread among various

warm-blooded animals, including domestic felines and humans(1). Approximately 1 billion people worldwide, including 22.5%of the population in the United States, are seropositive for T. gon-dii (2). Toxoplasmosis affects immunocompromised individuals,such as AIDS patients, organ transplant recipients, and the fetusesof newly infected mothers (2, 39–41). Although the incidence ofcongenital and AIDS-associated toxoplasmosis has been decreas-ing, recent waterborne outbreaks have shown that toxoplasmosisin immunocompetent individuals is more common than initiallyrealized. Unfortunately, current therapeutic options for toxoplas-mosis are limited. Antifolate drugs such as pyrimethamine areeffective against the tachyzoite stage of T. gondii but do not affectthe bradyzoite stage that causes chronic infection in the host.Lifelong maintenance with a combination of pyrimethamine-sul-fadiazine for toxoplasmic encephalitis often leads to side effects,including severe allergic reactions and hematotoxicity (3). Alter-native chemotherapeutic strategies are needed to prevent the on-set of these adverse reactions (4, 5).

Toxoplasma gondii is a member of the phylum Apicomplexa,which also includes Plasmodium, the causative agent of malaria.Both parasites replicate rapidly and require large amounts of pu-rines for the synthesis of their nucleic acids and other vital com-ponents. These obligate intracellular parasites cannot synthesizepurines de novo and depend on purine salvage from the host.Toxoplasma gondii nucleobase and nucleoside transporters havebeen identified and include TgNBT1, TgAT1, and TgAT2 (6–8).TgAT2 has an affinity for nucleosides, with a high affinity foradenosine (6, 9, 10). The rate of adenosine incorporation is higherthan that of other purines, and adenosine kinase (AdK) has a highlevel of activity relative to other enzymes in the pathway, indicat-ing that adenosine is the major purine utilized by T. gondii (9). Incontrast, Plasmodium falciparum has no AdK activity (11), and noAdK gene has been identified in the Plasmodium genome (12).However, in the presence of excess adenosine, P. falciparum can

use AMP synthesized by human erythrocyte AdK, which is fol-lowed by parasite uptake of AMP from the erythrocyte cytosol(11).

Toxoplasma can replicate normally in vitro using adenosinekinase or in the absence of adenosine kinase by using pathwaysthat require hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) activity (13). Toxoplasma gondii organ-isms with a �AdK background are viable, but genetic ablation ofAdK plus PNP inhibition kills the parasite (13). PNP convertsinosine to hypoxanthine and guanosine to guanine. TgPNP,which is structurally similar to P. falciparum PNP (PfPNP), has ahomohexameric structure, in contrast to homotrimeric mamma-lian PNP (14, 15). The structure and function of PfPNP is uniquebecause of its acceptance of 5=-methylthioinosine (MTI), a spe-cific metabolite of many Plasmodium species but one that is notpresent in the human host or in T. gondii (15, 16). The TgPNPamino acid sequence is 41% identical to that of PfPNP, and aprevious study showed that MTI is a poor substrate for TgPNP(13).

Immucillin-H (Imm-H) is a transition-state analogue inhibi-tor of PNP that causes purine starvation in cultured P. falciparum(17, 18) and inhibits both TgPNP and host cell PNP activity (13).To understand the structural basis of substrate and inhibitor rec-ognition, TgPNP was cocrystallized with immucillin-H. Thestructure of TgPNP in complex with immucillin-H and phosphate

Received 3 December 2013 Accepted 24 February 2014

Published ahead of print 28 February 2014

Address correspondence to Kami Kim, kami.kim@einstein.yu.edu.

T.M.D. and M.B.C. contributed equally to this study.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/EC.00308-13.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/EC.00308-13

572 ec.asm.org Eukaryotic Cell p. 572–579 May 2014 Volume 13 Number 5

on July 12, 2018 by guesthttp://ec.asm

.org/D

ownloaded from

revealed a reduced capacity to bind 5=-substituted nucleosides.Two representative 4=-deaza-1=-aza-2=-deoxy-1=-(9-methylene)(DADMe)-immucillins (19) were also tested. These analogueshave affinity similar to that of immucillin-H for PfPNP and ahigher affinity for human PNP (Fig. 1). Surprisingly, these ana-logues show low affinity for TgPNP. Several PNP family transi-tion-state analogues were used to explore catalytic site interac-tions of TgPNP (Fig. 1). The results indicate an early dissociativetransition state for TgPNP.

(Part of this work was published as part of a thesis submitted in

partial fulfillment of the requirements for a Ph.D. in BiomedicalSciences at the Albert Einstein College of Medicine [Teraya M.Donaldson].)

MATERIALS AND METHODSReagents. Xanthine oxidase, inosine, ampicillin, isopropyl �-D-1-thioga-lactopyranoside (IPTG), and protease inhibitor cocktail were purchasedfrom Sigma (St. Louis, MO). One Shot Top 10 chemically competentEscherichia coli cells, DNase I, Superscript III reverse transcriptase, Plati-num Taq high-fidelity master mix, and PtrcHis 2 Topo vectors were pur-

FIG 1 Structure of transition-state analogues used in this study to reveal differences in the TgPNP transition state and substrate specificity. The dissociationconstants for TgPNP (Tg), PfPNP (Pf), and HsPNP (Hs) were obtained using inosine as the substrate. Dissociation constant is defined as the tightest inhibitionconstant, either Ki or Ki*. An asterisk in parentheses indicates that the Ki* value is shown.

Toxoplasma gondii Purine Nucleoside Phosphorylase

May 2014 Volume 13 Number 5 ec.asm.org 573

on July 12, 2018 by guesthttp://ec.asm

.org/D

ownloaded from

chased from Invitrogen (Carlsbad, CA). BL21-codon plus (DE3)-RIPL E.coli competent cells were purchased from Stratagene (Santa Clara, CA).RNeasy minikits and nickel-nitrilotriacetic acid (Ni-NTA) agarose werepurchased from Qiagen (Valencia, CA). Imm-H, 5=-D-Imm-H, 5=-fluoro-Imm-H (5=-F-Imm-H), 5=-COOH-Imm-H, 2=-D-Imm-H, DADMe-Imm-H, DADMe-Imm-G, 5=-methylthio-Imm-H (5=-MT-Imm-H), 5=-CONH2-Imm-H, 5=-thio-Imm-H, and 1=,9-Me-Imm-H were synthesized asdescribed previously (15, 20, 21). Crystallography reagents and plates werepurchased from Hampton Research (Aliso Viejo, CA).

cDNA synthesis and PCR analysis of TgPNP. Toxoplasma gondii RHtachyzoite cDNA was synthesized from total cellular RNA, which wasprepared using chloroform-TRIzol (1:5, vol/vol). RNA was quantifiedusing a NanoDrop spectrophotometer and then treated with DNase I(RNase-free) at 37°C for 15 min prior to cDNA synthesis. RNA was puri-fied using a Qiagen RNeasy extraction kit according to the manufacturer’sprotocol. Aliquots containing 3.5 �g of RNA were stored at �80°C untilneeded. First-strand cDNA was generated using Invitrogen Superscript IIIreverse transcriptase and oligo(dT)20 as described by the manufacturer(22). PCR products from cDNA and genomic DNA (gDNA) were assessedon an agarose gel and analyzed via automated DNA sequencing (AlbertEinstein College of Medicine DNA Sequencing Facility, Bronx, NY).

Development of TgPNP constructs. The coding region of TgPNP,without the stop codon, was amplified by PCR using high-fidelity Taqwith the sense primer 5=-AGGGCATGGAAGTTCAGCCTC-3= and anti-sense primer 5=-GTACTGGCGACGCAGATTC-3=. The coding regionwas then cloned into the pTrcHis2-TOPO vector (Invitrogen) with a C-terminal hexahistidine tag and an ampicillin selection cassette. Each plas-mid was transformed into E. coli strain BL21-codon plus (DE3)-RIPL(Stratagene). The TgPNP clone used in this study contains a Val233Ilesubstitution not found in the TgPNP amino acid sequences reported ear-lier (13). This conservative substitution is remote from the catalytic siteand is not expected to cause significant differences in enzymatic activity. Itshould be noted that the genes for strains GT1 TGGT1_307030 and ME49TGME49_307030 found at the Toxoplasma Genomics Resource website,www.toxoDb.org, are incorrectly predicted to encode a 330-amino-acidprotein, in contrast to the 247-amino-acid protein previously character-ized (13) and predicted for the T. gondii VEG strain TGVEG_050700.

Expression and purification of TgPNP for kinetic studies. Fresh en-zyme was expressed and purified before each experiment. The recombi-nant enzyme was expressed by inducing a 100-ml bacterial culture with 1mM IPTG at 37°C for 18 h. Cells were ruptured by resuspension in Bug-Buster (Novagen, Darmstadt, Germany) according to the manufacturer’sinstructions, and the cell debris was removed by centrifugation at16,000 � g for 20 min at 4°C. Recombinant TgPNP was purified using aNi-NTA affinity chromatography spin column (Qiagen) according to themanufacturer’s instructions. Purified recombinant protein was buffer ex-changed against 50 mM Na2HPO4-KH2PO4 (pH 5.0), 1 mM DL-dithio-threitol (DTT), and 50 mM NaCl. This buffer condition was selected afterscreening (described below). The enzyme concentration was determinedfrom the extinction coefficient (24,005 M�1 cm�1) at 280 nm.

Buffer optimization for protein stability. Recombinant TgPNP wasexpressed and purified as described above. Immediately after purification,the recombinant protein was diluted 100-fold into 50 mM test buffercontaining 50 mM NaCl and 1 mM DTT (see Table S1 in the supplementalmaterial), and an aliquot from each condition was tested for PNP activityas described below. The remaining protein was incubated for 6 h at 4°Cand then retested for PNP activity.

Enzymatic assays and inhibition studies. Recombinant proteins wereused for enzymatic assays directly following purification. Kinetic assayswere carried out in 50 mM KH2PO4 at pH 7.4, measuring phosphorylysisof inosine by PNP in a coupled reaction with 60 mU/ml of xanthineoxidase to convert hypoxanthine to uric acid. Formation of uric acid wasmeasured at a wavelength of 293 nm (ε293 � 12.9 mM�1 cm�1) (17, 18).Assays were performed with excess substrate in the presence of inhibitors.Inhibition assays for determining the equilibrium dissociation constant

for the inhibitor (Ki) and for initial- and slow-onset inhibition constants(Ki*) were performed using various concentrations of Imm-H, 5=-D-Imm-H, 5=-F-Imm-H, 5=-COOH-Imm-H, 2=-D-Imm-H, DADMe-Imm-H, DADMe-Imm-G, 5=-MT-Imm-H, 5=-CONH2-Imm-H, 5=-thio-Imm-H, and 1=,9-Me-Imm-H, along with 500 �M inosine. Inhibitionconstants were determined as described previously (23). Initial-onset in-hibition was analyzed using the following equation: � � (kcat � [S])/{Km(1 [I]/Ki) [S]}, where � represents the steady-state rate, kcat

represents the catalytic rate, [S] represents the substrate concentration,Km represents the Michaelis constant for inosine, and [I] represents theinhibitor concentration. Human and P. falciparum PNPs were used ascontrols and were expressed and purified as described elsewhere (15, 16).

Protein crystallization and data collection. Bacterial cultures for ex-pressing TgPNP were grown in Luria Bertani-ampicillin broth at 37°C toan optical density at 595 nm of 0.6, induced with IPTG at a final concen-tration of 1 mM, and grown at 25°C for an additional 18 h. Cells wereharvested by centrifugation (4,000 � g for 30 min) and then ruptured bypassage through a French press. The resulting cell debris was pelleted bycentrifugation (16,000 � g for 30 min), and the remaining supernatantwas purified over a 3-ml Ni-NTA affinity column (Qiagen) with elutionby a step gradient of 50, 75, 100, 200, 300, and 500 mM imidazole in 50mM HEPES (pH 8.0), 300 mM NaCl, and 1 mM DTT. The purified re-combinant protein was dialyzed overnight against two different condi-tions: ammonium acetate buffer (50 mM ammonium acetate [pH 5.0], 50mM NaCl, and 1 mM DTT) and phosphate buffer (25 mM Na2HPO4-KH2PO4 [pH 5.0], 50 mM NaCl, and 1 mM DTT). The final concentra-tion of TgPNP for crystallization was 10 mg/ml in the presence of 3 mMImm-H and 10 mM phosphate. The crystallization condition of 25%polyethylene glycol monomethyl ether 2000, 100 mM Tris (pH 8.5), and0.2 M trimethylamine N-oxide dihydrate was determined using HamptonResearch Index HT screening by sitting-drop vapor diffusion. Crystalswere transferred into a fresh drop of the crystallization solution contain-ing 25% glycerol and rapidly frozen in liquid nitrogen. X-ray diffractiondata for Imm-H bound to TgPNP were collected using Beamline X29A atthe Brookhaven National Laboratory. All data were processed using theHKL2000 program suite, and the data-processing statistics are providedin Table 1 (24).

Structure determination and refinement. The crystal structure of Tg-PNP bound to immucillin-H was determined by molecular replacementin MolRep using the published structure of PfPNP bound to immucil-lin-H (PDB code 1NW4) as the search model, followed by model buildingusing Phenix (25, 26). The model without immucillin-H was first rebuiltusing the crystallographic object-oriented toolkit (COOT) and refined inRefmac5 (27, 28). Immucillin-H was added last using an Fo-Fc map andrefined in Refmac5 (28). The refinement statistics are summarized inTable 1.

PDB accession number. The coordinates and structure factors forImm-H-bound TgPNP were deposited in the Protein Data Bank (PDB)under accession code 3MB8.

RESULTSBuffer optimization for protein stability. Recombinant TgPNPenzyme activity was unstable, precluding previous attempts atcrystallization. Buffer composition and pH were optimized to sta-bilize the enzyme activity (see Table S1 in the supplemental ma-terial). With inosine as the substrate and Na2HPO4-KH2PO4 buf-fer at pH 5 and pH 6 or ammonium acetate buffer at pH 7, PNPactivity was retained (see Fig. S1).

Kinetic analysis of TgPNP. TgPNP accepts multiple sub-strates, including guanosine and inosine (13). Inosine is a purinesalvage metabolite and was used to establish kinetic parameters.The catalytic efficiency (kcat/Km) with inosine was 5.5 � 104 forTgPNP and 1.5 � 105 for PfPNP, similar to the values reportedpreviously for these enzymes (Table 2) (13, 15).

Donaldson et al.

574 ec.asm.org Eukaryotic Cell

on July 12, 2018 by guesthttp://ec.asm

.org/D

ownloaded from

Transition-state analogue screening. Transition-state ana-logues that mimic the specific geometry and electrostatic featuresof a molecule can be used to predict the position of the transitionstate in the reaction coordinates (29). The dissociation constantsof an array of transition-state analogue inhibitors were measuredto explore the specificity of TgPNP. Immucillin-H bound TgPNPtightly, with a Ki of 0.37 nM, similar to the Ki of 0.86 nM for PfPNP(Fig. 1; also see Table S2 in the supplemental material) (13).DADMe-Imm-H and DADMe-Imm-G are expanded-spectrumimmucillins and are powerful inhibitors of human PNP, with Ki*values of 0.0085 and 0.007 nM, respectively. These analogues in-hibited PfPNP with an affinity similar to that of Imm-H (Fig. 1;also see Table S2) (15). Surprisingly, these transition-state ana-logues bound poorly to TgPNP, with Ki values of 3,600 nM forDADMe-Imm-H and 1,500 nM for DADMe-Imm-G. BothImm-H (Ki � 0.37 nM) and Imm-G (Ki* � 1.9 nM) are powerfulinhibitors of TgPNP (13). Imm-H and Imm-G also bound PfPNPtightly, with Ki* values of 0.6 and 0.9 nM, respectively (15).DADMe-Imm-H and DADMe-Imm-G mimic a fully dissociatedSN1 ribocationic transition state.

High-affinity transition-state analogues for human PNP(HsPNP) were designed with a methylene bridge between the pu-rine and ribocation group, and these analogues lacked the 2=-

hydroxy group (Fig. 1) (30). The linear distance between thedeazapurine ring and the C1= of Imm-H was found to be 1.5 Å,characteristic of an early transition state, whereas the distance tothe N1= of DADMe-Imm-H was determined as 2.5 Å, which wascharacteristic of a late transition state (30). To evaluate if the pres-ence of the methylene bridge is responsible for the loss of affinity,1=,9-Me-Imm-H was tested with TgPNP. No inhibition was ob-served at 50 �M with 1=,9-Me-Imm-H, although 1=,9-Me-Imm-Hinhibited HsPNP with a Ki of 250 nM. These results indicate anearly dissociative transition state for TgPNP. In addition, 2=-D-Imm-H displayed a 522-fold decrease in affinity for TgPNP com-pared to Imm-H, indicating that the 2=-hydroxyl group is neces-sary for tight binding (Fig. 1; also see Table S2 in the supplementalmaterial). This specificity suggests a physiological role for TgPNPin nucleoside salvage. TgPNP did not show slow-onset inhibitionwith any inhibitor.

5=-MT-Imm-H was specifically designed to examine the sub-strate specificity of PfPNP for MTI. MTI is a poor substrate forTgPNP; thus, 5=-methylthio-purines are unlikely to be formedduring T. gondii metabolism (13). Consistent with this, 5=-MT-Imm-H was found to be a poor inhibitor of TgPNP, with a Ki of5,600 nM (Fig. 1; also see Table S2 in the supplemental material).The inhibitory activity of 5=-CONH2-Imm-H was similar to thatof 5=-MT-ImmH, with a Ki of 8,900 nM, and no inhibition wasobserved with 5=-thio-Imm-H at 50 �M. Other 5= modificationsrevealed a high selectivity for a hydroxyl group in the 5= position(Fig. 1; also see Table S2).

Crystal structure of TgPNP-bound Imm-H. The hexamericquaternary structure was similar to that of the Apicomplexa phy-lum member P. falciparum PNP and to E. coli PNP (Fig. 2A) butdiffered from that of mammalian trimeric family 1 PNP (15, 31).TgPNP cocrystallized in the presence of Imm-H showed a mono-mer structure consisting of a core containing 11 � sheets (Fig. 2B).TgPNP consists of a trimer of dimers, with six catalytic sitesformed by residues at the interface of the monomeric pairing (Fig.2A). Each subunit primarily houses one inhibitor molecule, withresidues His9 and Arg47 from the adjacent subunit binding tothe 5=-hydroxyl of Imm-H and the phosphate ion, respectively(Fig. 3).

Clear electron density at 1.8-Å resolution was observed for thetransition state analogue and phosphate in the active site of theenzyme. The deazahypoxanthine base was bound in a pocketformed by nonpolar residues Phe162, Pro210, Ile182, and Trp213and by polar residue Asp207. The N1= proton of the deazapurinehydrogen bonded with structurally conserved water at 2.8 Å,which was stabilized in a water lattice with another water moleculeand the backbone oxygen of Tyr161 (Fig. 4). Asp207 was observedto interact with the N7 proton of Imm-H at 3.0 Å. It formedwater-mediated hydrogen bonds with both O6 and O5= of the

TABLE 2 Kinetic constants for T. gondii, P. falciparum, and humanPNPs with inosine as the substrate

Source kcat (s�1) Km (�M) kcat/Km (M�1 s�1)

T. gondii 0.23 � 0.06 4 � 2 5.5 � 104

T. gondiia 2.6 � 0.0 13 � 1 2.0 � 105

P. falciparum 1.7 � 0.7 11 � 5 1.5 � 105

H. sapiensb 12 � 3 31 � 5 8.0 � 105

a Values originally reported in Chaudhary et al. (13).b Values originally reported in Lewandowicz et al. (30).

TABLE 1 Data collection and refinement statistics

Parameter Value(s) for Imm-H-bound TgPNPa

Data collectionSpace group P6Cell dimensions

a, b, c (Å) 159.6, 159.6, 53.6�, �, (°) 90.0, 90.0, 120.0

Resolution (Å) 20.00–1.90 (1.97–1.90)Rsym (%) 13.6 (54.1)I/�I 18.0 (2.9)Completeness (%) 94.4 (99.4)Redundancy 11.4 (7.0)

Refinement statisticsResolution range (Å) 20.0–1.9No. of reflections 61,967Rwork/Rfree (%) 22.1/24.3

B factors (Å2)Protein (main chain) 17.2Protein (side chain) 19.5Water 31.7Ligand 12.4

No. of atomsProtein 3,770Water 340Ligand 48

RMSDBond length (Å) 0.012Bond angle (°) 1.46

Ramachandran plot (%)Allowed region 100.0Disallowed region 0.0

a Numbers in parentheses are for the highest-resolution shell. One crystal was used foreach data set. The PDB code for Imm-H-bound TgPNP is 3MB8.

Toxoplasma gondii Purine Nucleoside Phosphorylase

May 2014 Volume 13 Number 5 ec.asm.org 575

on July 12, 2018 by guesthttp://ec.asm

.org/D

ownloaded from

inhibitor and is favored to be the general acid/base for protona-tion of N7 with formation of the transition state (15, 32, 33).

Tyr218 formed a water-mediated interaction with the 5=-hy-droxyl of the iminoribitol group (2.7 Å) and formed hydrogen

bonds with the O2 of a glycerol molecule (2.6 Å) from the crystal-lization buffer in the active site. His9 NE2 from the adjacent sub-unit was within hydrogen bonding distance to the 5=-hydroxyl(2.7 Å). Glu185 was bound to both the 2=- and 3=-hydroxyls of theiminoribitol group. The phosphate ion was stabilized in an an-ionic pocket consisting of Arg90, Arg47b, Arg29, and the �-polarresidue Thr93 beneath the inhibitor. The OG1 of Thr93 waswithin hydrogen bonding distance to the N1= of the ribitol group(3.2 Å). Ion pairing to the phosphate ion occurred with residues

FIG 2 (A) X-ray crystal structure of the TgPNP hexamer with immucillin-Hand PO4

3� bound in the active site. The hexamer structure was generated byapplying crystallographic symmetry to the dimers composed of the light blueand dark blue subunits on the bottom left, yielding the purple, green, yellow,and orange monomers. (B) TgPNP monomer with immucillin-H and PO4

3�

bound in the active site (in yellow). The monomer consists of eight � helices inlight blue and surrounds 11 � sheets in the red subunit. Loop regions areshaded in purple. The figure was prepared with MacPyMol.

FIG 3 Stereo views of the catalytic site contacts in TgPNP with the transition-state analogue inhibitor immucillin-H and PO43�. The structure shows light blue

side chains of the parental monomer surrounding the bound Imm-H (yellow); the green subunits on the bottom right indicate residues contributed from theadjacent subunit. Residues participating in binding Imm-H and PO4

3� are labeled in the active site of TgPNP. The figure was prepared with MacPyMol.

FIG 4 Schematic diagram of catalytic site contacts for immucillin-H andPO4

3� at the active site of TgPNP. Amino acids are from the parent subunitunless labeled with a b, which marks residues from the adjacent subunit.Dashed lines indicate hydrogen bonding. Distances are shown in angstroms.

Donaldson et al.

576 ec.asm.org Eukaryotic Cell

on July 12, 2018 by guesthttp://ec.asm

.org/D

ownloaded from

Arg90 NH1 and NH2 (3.1 to 2.8 Å), Arg47b NH1 and NH2 (2.9 to3.0 Å), and Arg29, with distances of 2.7 Å for both NH1 and NH2.

Comparison of the active sites of TgPNP and PfPNP. In thestructures depicted in Fig. 5, residues His9, Ile68, Ile75, Phe162,and Tyr218 are shown to illustrate the similarities found in theactive sites of TgPNP to those of PfPNP bound to Imm-H (PDBcode 1NW4). The two homologous enzymes shared 41% se-quence identity, and their C� backbone superimposed well, withroot mean square deviations (RMSD) of 0.7 Å out of 1,208 atomsfor 1NW4 and 0.7 Å out of 1,220 atoms for 1Q1G. Even though thesequence identity was only 41%, with the exception of Ile68, Ile75,Phe162, and Thr93, there was a high degree of identity with re-spect to the active pocket residues between the two isozymes. Thecorresponding residues for PfPNP were Val66, Val73, Tyr160, andSer91. The first three residues are proposed to play a key role inbinding the 5=-methylthio group in the pocket (15). A water mol-ecule was found to be conserved between TgPNP-bound Imm-Hand PfPNP-bound Imm-H (1NW4). Both Ile68 and Ile75 werefound to carry an additional methyl group compared to Val66 andVal73. These bulky groups protrude an additional 1.5 Å into theactive site, a distance of 4.1 Å from C5= and 5.0 Å from O5= ofImm-H in the crystal structure of 1NW4. When 5=-MT-Imm-H-bound PfPNP (PDB code 1Q1G) was overlaid with Imm-H-bound TgPNP, Val66 and Val73 were found to be within Van derWaals distances of 2.5 and 1.8 Å from Ile68 and Ile75, creating asteric clash (Fig. 5B). The 5=-hydroxyl and 5=-methylthio groupsof Imm-H and 5=-MT-Imm-H were in different orientations. InTgPNP-bound Imm-H, the 5=-hydroxyl was in a position that issurrounded by hydrophilic residues in a solvent-filled cavity,whereas PfPNP-bound 5=-MT-Imm-H (1Q1G) was observed tohave the 5=-methylthio group rotated 135° away, toward a closedhydrophobic cavity encompassed by Val66, Val73, and Tyr160(15).

DISCUSSIONImprovement of TgPNP stability after purification. The stabilityof TgPNP was improved by buffer screening. The kcat/Km was sim-ilar to that of earlier reports, but the stabilized protein had a lowerkcat (Table 2) (13). Buffer screening enabled the synthesis of viableTgPNP crystals for crystallographic structural determination andinhibition studies.

Transition-state analogue screening. Transition-state struc-tures can be predicted based upon affinity to related transition-state analogues. The transition-state characteristics are comparedby analyzing dissociation constants (Ki) of an array of representa-tive transition-state analogues (29). These inhibitors mimicknown early or late dissociative transition states. Transition-statestructures of N-ribosyl phosphorylases that metabolize nucleo-sides can be either early or late dissociative and are distinguishedby the distance between the ribosyl anomeric carbon and the N9 ofthe purine base-leaving group (23). The narrow-spectrum PNPtransition-state analogue immucillin-H was designed from thetransition state of bovine PNP, which revealed an early transitionstate with ribocation characteristics but a relatively close 1.8-Ådistance between the leaving-group nitrogen and the anomericcarbon (34). In contrast, HsPNP has a fully dissociated purineleaving group with a fully developed ribocation, with a distancegreater than 2.6 Å between the leaving-group nitrogen and theanomeric carbon (30). An expanded-spectrum HsPNP transition-state analogue inhibitor, DADMe-Imm-H, was designed to matchthis characteristic by the addition of a methylene bridge betweenthe purine and the sugar moiety (19) (Fig. 1). TgPNP was stronglyinhibited by immucillin-H, with a Ki of 0.37 nM. Surprisingly,TgPNP was weakly inhibited by DADMe-Imm-H and DADMe-Imm-G, with Ki values of 3,600 and 1,500 nM, respectively, indi-cating an early dissociative transition state for TgPNP. As shownin Fig. 1, no inhibition was observed with 1=,9-Me-Imm-H at 50�M, which also supports the formation of an early transition state.Despite the high degree of residue conservation found in the activesites of HsPNP and TgPNP, inhibitor specificity studies supportdistinct transition states. Moreover, different affinities of PfPNPand TgPNP for the analogues tested indicate divergence of thetransition states among Apicomplexa PNPs.

TgPNP substrate specificity. Consistent with the observationthat TgPNP exhibits substrate specificity for inosine but not forMTI, TgPNP inhibition assays with immucillin-H and 5=-MT-Imm-H yielded Ki values of 0.37 and 5,600 nM, respectively. Be-cause the 5=-CONH2 group is able to hydrogen bond with His9,5=-CONH2-Imm-H was tested as an alternative transition-stateanalogue to the 5=-methylthio group of 5=-MT-Imm-H. However,5=-CONH2-Imm-H showed weak binding, with a Ki of 8,900 nM,suggesting that this 5= group is too bulky. For both 5=-MT-Imm-Hand 5=-CONH2-Imm-H, the weak binding reflects space con-straints in the 5=-group binding cavity. This restriction at the 5=position is supported by the results of tests involving several dis-tinct inhibitors with specific 5=modifications (Fig. 1; also see Ta-ble S2 in the supplemental material). TgPNP exhibits a high de-gree of selectivity for a hydroxyl group in the 5= position, asevidenced by results showing that all 5=modifications resulted in adecrease in binding affinity compared with Imm-H (Imm-H �5=-D-Imm-H � 5=-F-Imm-H � 5=-COOH-Imm-H � 5=-MT-Imm-H � 5=-CONH2-Imm-H � 5=-thio-Imm-H).

TgPNP crystal structure. The residues in the active sites of

FIG 5 (A) Cross-eyed stereo-view superposition of TgPNP Imm-H-PO43�

(purple; PDB code 3MB8) and PfPNP Imm-H-SO4 (light blue; PDB code1NW4) to show differences in amino acids surrounding the inhibitor. (B)Cross-eyed stereo-view superposition of TgPNP Imm-H-PO4

3� (purple; PDBcode 3MB8) and PfPNP 5=-MT-Imm-H-SO4 (yellow; PDB code 1Q1G) toshow the relative angular shifting of residues in the active site when 5=-MT-Imm-H is bound.

Toxoplasma gondii Purine Nucleoside Phosphorylase

May 2014 Volume 13 Number 5 ec.asm.org 577

on July 12, 2018 by guesthttp://ec.asm

.org/D

ownloaded from

TgPNP and PfPNP are highly conserved, with the exception ofIle68, Ile75, Phe162, and Thr93 in TgPNP, which correspond toVal66, Val73, Tyr160, and Ser91 in PfPNP. The first three residuesare proposed to play a key role in binding the 5=-methylthio groupin the pocket (15). It was found previously that PfPNP Tyr160 wasable to form two water-mediated hydrogen bonds with O5= andAsp206, but TgPNP Phe162 does not participate in hydrogenbonding to water in the pocket according to our experiments.Residue Tyr218, which is located within the TgPNP active site,makes conserved water-mediated contacts that substitute forTyr160 in PfPNP.

In the structure of PfPNP bound to 5=-MT-Imm-H, the 5=-methylthio group is turned 135° relative to the 5=-hydroxyl groupof immucillin-H in TgPNP Imm-H, abutting residues in a denselypacked hydrophobic area in the catalytic site. The 5=-hydroxylgroup of immucillin-H is surrounded by largely hydrophilic resi-dues within a solvent-filled cavity in PfPNP. These findings agreewith reported positions of both immucillin-H and 5=-MT-Imm-H bound to PfPNP (15).

T. gondii and polyamine salvage. Similar to other members ofthe phylum Apicomplexa, such as Plasmodium and Cryptospo-ridium, T. gondii is a purine auxotroph that relies on purine sal-vage for its metabolic requirements (35, 36). The difference in thespecificities of TgPNP and PfPNP relates to distinct metabolicfunctions within the respective parasites. Toxoplasma gondii livesin nucleated mammalian cells. As polyamines are abundant inthese cells, it is believed that polyamine salvage replaces de novopolyamine synthesis (37, 38). Under conditions of polyamineabundance, no 5=-methylthioadenosine, a by-product of poly-amine synthesis, is formed in T. gondii. In contrast, P. falciparummust synthesize polyamines because erythrocytes are polyaminedeficient. The resulting 5=-methylthioadenosine formed is metab-olized by unique dual-specificity N-(2-acetamido)iminodiaceticacid and PNP enzymes in most Plasmodium species, hence thespecificity for 5=-methylthioinosine in PfPNP.

Conclusions. Due to a lack of forward-directed polyaminebiosynthetic enzymes, T. gondii is unable to generate 5=-methyl-thiopurines (37, 38). Examination of the structure of TgPNP in-dicates that the residues in the active site cannot accommodate5=-methylthioinosine, but the solvent-filled pocket surroundingthe 5=-OH group of immucillin-H suggests that the active site isable to accept a larger 5= group. Inhibitor screening of 5= analoguesrevealed a high selectivity for a hydroxyl group in the 5= position.Any modification resulted in a decrease in binding affinitycompared with binding to immucillin-H. Moreover, the resultspresented here show that TgPNP is strongly inhibited by immu-cillin-H but weakly inhibited by DADMe-Imm-H and DADMe-Imm-G. In addition, no inhibition was detected with 1=,9-Me-Imm-H, indicating an early dissociative transition state for thisenzyme. This information reveals a surprising degree of catalyticdivergence among Apicomplexa PNPs.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health (NIH) researchgrant R01AI049512 (to V.L.S.).

Data for this study were measured at Beamline X29 of the NationalSynchrotron Light Source. Financial support comes principally from theOffices of Biological and Environmental Research and of Basic EnergySciences of the U.S. Department of Energy, the National Center for Re-

search Resources (P41RR012408), and the National Institute of GeneralMedical Sciences (P41GM103473) of the National Institutes of Health.

REFERENCES1. Kim K, Weiss LM. 2004. Toxoplasma gondii: the model apicomplexan.

Int. J. Parasitol. 34:423– 432. http://dx.doi.org/10.1016/j.ijpara.2003.12.009.

2. Jones JL, Kruszon-Moran D, Wilson M, McQuillan G, Navin T, Mc-Auley JB. 2001. Toxoplasma gondii infection in the United States: sero-prevalence and risk factors. Am. J. Epidemiol. 154:357–365. http://dx.doi.org/10.1093/aje/154.4.357.

3. Al Safarjalani ON, Rais RH, Kim YA, Chu CK, Naguib FN, el KouniMH. 2008. 7-Deaza-6-benzylthioinosine analogues as subversive sub-strate of Toxoplasma gondii adenosine kinase: activities and selective tox-icities. Biochem. Pharmacol. 76:958 –966. http://dx.doi.org/10.1016/j.bcp.2008.07.035.

4. Bosch-Driessen LH, Verbraak FD, Suttorp-Schulten MS, van RuyvenRL, Klok AM, Hoyng CB, Rothova A. 2002. A prospective, randomizedtrial of pyrimethamine and azithromycin vs pyrimethamine and sulfadi-azine for the treatment of ocular toxoplasmosis. Am. J. Ophthalmol. 134:34 – 40. http://dx.doi.org/10.1016/S0002-9394(02)01537-4.

5. Donald RG, Roos DS. 1993. Stable molecular transformation of Toxoplasmagondii: a selectable dihydrofolate reductase-thymidylate synthase marker basedon drug-resistance mutations in malaria. Proc. Natl. Acad. Sci. U. S. A. 90:11703–11707. http://dx.doi.org/10.1073/pnas.90.24.11703.

6. De Koning HP, Al-Salabi MI, Cohen AM, Coombs GH, Wastling JM.2003. Identification and characterisation of high affinity nucleoside andnucleobase transporters in Toxoplasma gondii. Int. J. Parasitol. 33:821–831. http://dx.doi.org/10.1016/S0020-7519(03)00091-2.

7. Chiang CW, Carter N, Sullivan WJ, Jr, Donald RG, Roos DS, NaguibFN, el Kouni MH, Ullman B, Wilson CM. 1999. The adenosine trans-porter of Toxoplasma gondii. Identification by insertional mutagenesis,cloning, and recombinant expression. J. Biol. Chem. 274:35255–35261.

8. Schwab JC, Afifi Afifi M, Pizzorno G, Handschumacher RE, Joiner KA.1995. Toxoplasma gondii tachyzoites possess an unusual plasma mem-brane adenosine transporter. Mol. Biochem. Parasitol. 70:59 – 69. http://dx.doi.org/10.1016/0166-6851(95)00005-L.

9. Krug EC, Marr JJ, Berens RL. 1989. Purine metabolism in Toxoplasmagondii. J. Biol. Chem. 264:10601–10607.

10. Pfefferkorn ER, Pfefferkorn LC. 1977. Toxoplasma gondii: specific label-ing of nucleic acids of intracellular parasites in Lesch-Nyhan cells. Exp.Parasitol. 41:95–104. http://dx.doi.org/10.1016/0014-4894(77)90134-5.

11. Cassera MB, Hazleton KZ, Riegelhaupt PM, Merino EF, Luo M, AkabasMH, Schramm VL. 2008. Erythrocytic adenosine monophosphate as analternative purine source in Plasmodium falciparum. J. Biol. Chem. 283:32889 –32899. http://dx.doi.org/10.1074/jbc.M804497200.

12. Gardner MJ, Hall N, Fung E, White O, Berriman M, Hyman RW,Carlton JM, Pain A, Nelson KE, Bowman S, Paulsen IT, James K, EisenJA, Rutherford K, Salzberg SL, Craig A, Kyes S, Chan MS, Nene V,Shallom SJ, Suh B, Peterson J, Angiuoli S, Pertea M, Allen J, SelengutJ, Haft D, Mather MW, Vaidya AB, Martin DM, Fairlamb AH, Fraun-holz MJ, Roos DS, Ralph SA, McFadden GI, Cummings LM, Subrama-nian GM, Mungall C, Venter JC, Carucci DJ, Hoffman SL, Newbold C,Davis RW, Fraser CM, Barrell B. 2002. Genome sequence of the humanmalaria parasite Plasmodium falciparum. Nature 419:498 –511. http://dx.doi.org/10.1038/nature01097.

13. Chaudhary K, Ting LM, Kim K, Roos DS. 2006. Toxoplasma gondii purinenucleoside phosphorylase biochemical characterization, inhibitor profiles, andcomparison with the Plasmodium falciparum ortholog. J. Biol. Chem. 281:25652–25658. http://dx.doi.org/10.1074/jbc.M602624200.

14. Ealick SE, Rule SA, Carter DC, Greenhough TJ, Babu YS, Cook WJ,Habash J, Helliwell JR, Stoeckler JD, Parks RE, Jr. 1990. Three-dimensional structure of human erythrocytic purine nucleoside phos-phorylase at 3.2 A resolution. J. Biol. Chem. 265:1812–1820.

15. Shi W, Ting LM, Kicska GA, Lewandowicz A, Tyler PC, Evans GB,Furneaux RH, Kim K, Almo SC, Schramm VL. 2004. Plasmodiumfalciparum purine nucleoside phosphorylase: crystal structures, immucil-lin inhibitors, and dual catalytic function. J. Biol. Chem. 279:18103–18106. http://dx.doi.org/10.1074/jbc.C400068200.

16. Ting LM, Shi W, Lewandowicz A, Singh V, Mwakingwe A, Birck MR,Ringia EA, Bench G, Madrid DC, Tyler PC, Evans GB, Furneaux RH,Schramm VL, Kim K. 2005. Targeting a novel Plasmodium falciparum

Donaldson et al.

578 ec.asm.org Eukaryotic Cell

on July 12, 2018 by guesthttp://ec.asm

.org/D

ownloaded from

purine recycling pathway with specific immucillins. J. Biol. Chem. 280:9547–9554. http://dx.doi.org/10.1074/jbc.M412693200.

17. Kicska GA, Tyler PC, Evans GB, Furneaux RH, Kim K, Schramm VL.2002. Transition state analogue inhibitors of purine nucleoside phosphor-ylase from Plasmodium falciparum. J. Biol. Chem. 277:3219 –3225. http://dx.doi.org/10.1074/jbc.M105905200.

18. Kicska GA, Tyler PC, Evans GB, Furneaux RH, Schramm VL, Kim K.2002. Purine-less death in Plasmodium falciparum induced by immucil-lin-H, a transition state analogue of purine nucleoside phosphorylase. J.Biol. Chem. 277:3226 –3231. http://dx.doi.org/10.1074/jbc.M105906200.

19. Evans GB, Furneaux RH, Lewandowicz A, Schramm VL, Tyler PC.2003. Synthesis of second-generation transition state analogues of humanpurine nucleoside phosphorylase. J. Med. Chem. 46:5271–5276. http://dx.doi.org/10.1021/jm030305z.

20. Evans GB, Furneaux RH, Schramm VL, Singh V, Tyler PC. 2004.Targeting the polyamine pathway with transition-state analogue inhibi-tors of 5=-methylthioadenosine phosphorylase. J. Med. Chem. 47:3275–3281. http://dx.doi.org/10.1021/jm0306475.

21. Lewandowicz A, Ringia EA, Ting LM, Kim K, Tyler PC, Evans GB,Zubkova OV, Mee S, Painter GF, Lenz DH, Furneaux RH, SchrammVL. 2005. Energetic mapping of transition state analogue interactionswith human and Plasmodium falciparum purine nucleoside phosphory-lases. J. Biol. Chem. 280:30320 –30328. http://dx.doi.org/10.1074/jbc.M505033200.

22. Stratmann U, Schaarschmidt K, Lehmann RR, Schurenberg M, WillitalGH, Berens A. 1995. The morphological tissue response of the pigletoesophagus to experimental irradiation by 1320 nm Nd:YAG laser. J.Anat. 187(Part 3):661– 670.

23. Lewandowicz A, Tyler PC, Evans GB, Furneaux RH, Schramm VL.2003. Achieving the ultimate physiological goal in transition state ana-logue inhibitors for purine nucleoside phosphorylase. J. Biol. Chem. 278:31465–31468. http://dx.doi.org/10.1074/jbc.C300259200.

24. Otwinowski Z, Minor W. 1997. Processing of X-ray diffraction datacollected in oscillation mode. Methods Enzymol. 276:307–326. http://dx.doi.org/10.1016/S0076-6879(97)76066-X.

25. Bailey S. 1994. The Ccp4 suite–programs for protein crystallography. Acta Crys-tallogr. D. 50:760–763. http://dx.doi.org/10.1107/S0907444994003112.

26. Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N,Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW, McCoy AJ,Moriarty NW, Oeffner R, Read RJ, Richardson DC, Richardson JS,Terwilliger TC, Zwart PH. 2010. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D.66:213–221. http://dx.doi.org/10.1107/S0907444909052925.

27. Emsley P, Cowtan K. 2004. Coot: model-building tools for moleculargraphics. Acta Crystallogr. D. 60:2126 –2132. http://dx.doi.org/10.1107/S0907444904019158.

28. Potterton E, Briggs P, Turkenburg M, Dodson E. 2003. A graphical userinterface to the CCP4 program suite. Acta Crystallogr. D. 59:1131–1137.http://dx.doi.org/10.1107/S0907444903008126.

29. Gutierrez JA, Luo M, Singh V, Li L, Brown RL, Norris GE, Evans GB,Furneaux RH, Tyler PC, Painter GF, Lenz DH, Schramm VL. 2007.Picomolar inhibitors as transition-state probes of 5=-methylthioadenosinenucleosidases. ACS Chem. Biol. 2:725–734. http://dx.doi.org/10.1021/cb700166z.

30. Lewandowicz A, Schramm VL. 2004. Transition state analysis for humanand Plasmodium falciparum purine nucleoside phosphorylases. Bio-chemistry 43:1458 –1468. http://dx.doi.org/10.1021/bi0359123.

31. Pugmire MJ, Ealick SE. 2002. Structural analyses reveal two distinctfamilies of nucleoside phosphorylases. Biochem. J. 361:1–25. http://dx.doi.org/10.1042/0264-6021:3610001.

32. Rinaldo-Matthis A, Wing C, Ghanem M, Deng H, Wu P, Gupta A,Tyler PC, Evans GB, Furneaux RH, Almo SC, Wang CC, Schramm VL.2007. Inhibition and structure of Trichomonas vaginalis purine nucleo-side phosphorylase with picomolar transition state analogues. Biochem-istry 46:659 – 668. http://dx.doi.org/10.1021/bi061515r.

33. Taylor EA, Rinaldo-Matthis A, Li L, Ghanem M, Hazleton KZ, CasseraMB, Almo SC, Schramm VL. 2007. Anopheles gambiae purine nucleo-side phosphorylase: catalysis, structure, and inhibition. Biochemistry 46:12405–12415. http://dx.doi.org/10.1021/bi7010256.

34. Kline PC, Schramm VL. 1993. Purine nucleoside phosphorylase. Cata-lytic mechanism and transition-state analysis of the arsenolysis reaction.Biochemistry 32:13212–13219.

35. Chaudhary K, Darling JA, Fohl LM, Sullivan WJ, Jr, Donald RG,Pfefferkorn ER, Ullman B, Roos DS. 2004. Purine salvage pathways inthe apicomplexan parasite Toxoplasma gondii. J. Biol. Chem. 279:31221–31227. http://dx.doi.org/10.1074/jbc.M404232200.

36. Abrahamsen MS, Templeton TJ, Enomoto S, Abrahante JE, Zhu G,Lancto CA, Deng M, Liu C, Widmer G, Tzipori S, Buck GA, Xu P,Bankier AT, Dear PH, Konfortov BA, Spriggs HF, Iyer L, Ananthara-man V, Aravind L, Kapur V. 2004. Complete genome sequence of theapicomplexan, Cryptosporidium parvum. Science 304:441– 445. http://dx.doi.org/10.1126/science.1094786.

37. Cook T, Roos D, Morada M, Zhu G, Keithly JS, Feagin JE, Wu G, Yarlett N.2007. Divergent polyamine metabolism in the Apicomplexa. Microbiology 153:1123–1130. http://dx.doi.org/10.1099/mic.0.2006/001768-0.

38. Seabra SH, DaMatta RA, de Mello FG, de Souza W. 2004. Endogenouspolyamine levels in macrophages is sufficient to support growth of Toxo-plasma gondii. J. Parasitol. 90:455– 460. http://dx.doi.org/10.1645/GE-179R.

39. Piazza E, Condorelli A, Arcidiacono R, Tropea R, Chiaramonte I, Mancuso P.1986. Intracerebral mass lesions in patients affected by AIDS. Acta Neurochir.(Vienna) 83:116–120. http://dx.doi.org/10.1007/BF01402389.

40. Levy RM, Pons VG, Rosenblum ML. 1984. Central nervous system masslesions in the acquired immunodeficiency syndrome (AIDS). J. Neuro-surg. 61:9 –16. http://dx.doi.org/10.3171/jns.1984.61.1.0009.

41. Kravetz JD, Federman DG. 2005. Toxoplasmosis in pregnancy. Am. J.Med. 118:212–216. http://dx.doi.org/10.1016/j.amjmed.2004.08.023.

Toxoplasma gondii Purine Nucleoside Phosphorylase

May 2014 Volume 13 Number 5 ec.asm.org 579

on July 12, 2018 by guesthttp://ec.asm

.org/D

ownloaded from